Nucleated Red Blood Cell Analysis System and Method

ABSTRACT

Systems and methods for analyzing blood samples, and more specifically for performing a nucleated red blood cell (nRBC) analysis. The systems and methods screen a blood sample by means of fluorescence staining and a fluorescence triggering strategy, to identify nuclei-containing particles within the blood sample. As such, interference from unlysed red blood cells (RBCs) and fragments of lysed RBCs is substantially eliminated. The systems and methods also enable development of relatively milder reagent(s), suitable for assays of samples containing fragile white blood cells (WBCs). In one embodiment, the systems and methods include: (a) staining a blood sample with an exclusive cell membrane permeable fluorescent dye; (b) using a fluorescence trigger to screen the blood sample for nuclei-containing particles; and (c) using measurements of light scatter and fluorescence emission to distinguish nRBCs from WBCs.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Patent Application No. 61/482,545, titled, Method ForAnalyzing Nucleated Red Blood Cells, and filed on May 4, 2011, theentire disclosure of which is incorporated by reference herein.

This application is also related to application Ser. No. ______, filedon Apr. 26, 2012, titled “WHITE BLOOD CELL ANALYSIS SYSTEM AND METHOD,”with Atty Dkt No: ADDV-016 (11040USO1); and application Ser. No. ______,filed on Apr. 26, 2012, titled “BASOPHIL ANALYSIS SYSTEM AND METHOD,”with Atty Dkt No: ADDV-018 (11042USO1), the entire disclosures of whichare herein incorporated by reference in their entirety.

BACKGROUND

This invention relates to hematology systems and methods. Morespecifically, this invention relates to systems and methods foranalyzing blood samples to identify, classify, and/or quantify nucleatedred blood cells (nRBCs) in a sample of blood.

Nucleated red blood cells are often present in the fetus and in theperipheral blood of newborns. However, nRBCs are considered to beabnormal for adults. The presence of nRBCs in an adult's peripheralblood stream is usually an indication of serious marrow stress. Studieshave shown that the appearance of nRBCs in the blood stream is highlycorrelated with severe disease stages and/or poor prognosis forcritically ill patients. Therefore, accurate identification andquantification of nRBCs has become increasingly important for clinicaldiagnostics.

Because nRBCs share numerous similarities with white blood cells (WBCs),the concentration of nRBCs in a blood sample is typically reported as apercentage of total WBCs in the blood sample (i.e., %nRBC=nRBCs/WBCs×100%). Traditional approaches to analyze nRBCs include:(1) separating nRBCs from WBCs by size; (2) differentiating nRBCs fromWBCs by means of light scattering; or (3) analyzing nRBCs by means offluorescence emission detection after lysis and staining with a cellmembrane impermeable fluorescent dye(s).

Each of the above-listed techniques has shown weaknesses in clinicalpractices. For example, it is difficult to completely eliminatefragments of lysed red blood cells (RBCs) in rapid hematologymeasurements. Because fragments of RBCs and the nuclei of nRBCs may besimilar in size and light scattering characteristics, analysis based onsize and/or light scattering is sometimes misleading. Meanwhile,analysis based on fluorescence emission may be adversely affected by:(1) “under-lysing” of the sample such that the cell membrane impermeabledye cannot reach the nuclei of the nRBCs; (2) “over-lysing” of thesample such that nuclei of the WBCs are stained and interfere with thenRBC count; (3) the existence of fragile lymphocytes, such that WBCs areunexpectedly hyper-sensitive to a lysing reagent (giving falsepositives); and/or (4) the existence of lyse-resistant nRBCs, such thatthe nRBCs are unexpectedly insensitive to a lysing reagent (giving falsenegatives). In practice, over-lysing or under-lysing is common onaccount of the variation in membrane rigidity of blood cells amongsamples of blood. As such, dependence on known light scatter and/orfluorescence emission detection techniques may result in an inaccurateand unreliable analysis for nRBCs, thereby preventing correct diagnosesand treatment for critically ill patients.

BRIEF SUMMARY

Provided herein are systems and methods for analyzing blood samples, andmore specifically for performing a nRBC analysis. The systems andmethods screen a blood sample by means of fluorescence staining with acell membrane permeable fluorescent dye. A fluorescence triggeringstrategy is then used to identify, distinguish, and separatenuclei-containing particles (e.g., nRBCs and WBC) fromnon-nuclei-containing particles (e.g., RBCs and/or RBC fragments) withinthe blood sample. As such, interference from unlysed RBCs and fragmentsof lysed RBCs can be substantially eliminated prior to subsequentanalysis. For example, in one embodiment, the systems and methodsinclude: (a) staining a blood sample with an exclusive cell membranepermeable fluorescent dye; (b) using a fluorescence trigger to screenthe blood sample for nuclei-containing particles; and then (c) usingmeasurements of light scatter and fluorescence emission to distinguishnRBCs from WBCs. The systems and methods enable development ofrelatively milder reagent(s), suitable for assays of samples containingfragile WBCs.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying drawings, which are incorporated herein, form part ofthe specification. Together with this written description, the drawingsfurther serve to explain the principles of, and to enable a personskilled in the relevant art(s), to make and use the systems and methodspresented. In the drawings, like reference numbers indicate identical orfunctionally similar elements.

FIGS. 1A-1D show histograms of a sample of whole blood, showing WBCs,nRBCs, and residues of RBCs following lysis.

FIG. 1A is a histogram showing measurements of axial light loss (ALL).

FIG. 1B is a histogram showing measurements of intermediate anglescatter (IAS).

FIG. 1C is a histogram showing measurements of 90° polarized sidescatter (PSS).

FIG. 1D is a histogram showing measurements of fluorescence (FL1).

FIG. 2 is a schematic diagram illustrating a hematology instrument.

FIGS. 3A-3F are cytograms illustrating an analysis of a sample of bloodcontaining nRBCs at a % NRBC of 75%.

FIG. 3A is a cytogram depicting axial light loss vs. intermediate anglescatter.

FIG. 3B is a cytogram depicting 90° polarized side scatter vs. axiallight loss.

FIG. 3C is a cytogram depicting 90° polarized side scatter vs.intermediate angle scatter.

FIG. 3D is a cytogram depicting FL1 vs. axial light loss.

FIG. 3E is a cytogram depicting FL1 vs. intermediate angle scatter.

FIG. 3F is a cytogram depicting FL1 vs. 90° polarized side scatter.

FIGS. 4A-4F are cytograms illustrating an analysis of a sample of bloodcontaining nRBCs as a % NRBC of 1.0%.

FIG. 4A is a cytogram depicting axial light loss vs. intermediate anglescatter.

FIG. 4B is a cytogram depicting 90° polarized side scatter vs. axiallight loss.

FIG. 4C is a cytogram depicting 90° polarized side scatter vs.intermediate angle scatter.

FIG. 4D is a cytogram depicting FL1 vs. axial light loss.

FIG. 4E is a cytogram depicting FL1 vs. intermediate angle scatter.

FIG. 4F is a cytogram depicting FL1 vs. 90° polarized side scatter.

FIG. 5 is a plot illustrating correlation of % NRBC as determined bymanual gating vs.

reference results, wherein the reference results were obtained by amicroscope review.

FIG. 6 is a plot illustrating correlation of % NRBC as determined bymanual gating vs.

reference results, wherein the reference results were obtained by a“CELL-DYN” Sapphire™ hematology analyzer.

FIG. 7 is a plot illustrating correlation of %NRBC, as determined byauto-clustering vs. reference results, wherein the reference resultswere obtained by a microscope review.

FIG. 8 is a plot illustrating correlation of %NRBC, as determined byauto-clustering vs. reference results, wherein the reference resultswere obtained by a “CELL-DYN” Sapphire™ hematology analyzer.

DETAILED DESCRIPTION

Provided herein are systems and methods for analyzing blood samples, andmore specifically for performing an nRBC analysis to identify, classify,and count nRBCs in a blood sample. In general, the systems and methodsdisclosed screen nuclei-containing events vs. non-nuclei-containingevents by means of fluorescence staining and a fluorescence triggeringstrategy. As such, interference from unlysed red blood cells (RBCs),such as lysis-resistant red blood cells (rstRBCs), and RBC fragments issubstantially eliminated prior to subsequent analysis. In other words,the systems and methods described herein utilize at least onefluorescent dye and a fluorescence triggering system to screen eventscontaining nuclei, to thereby accurately and reliably identify andquantify WBCs and nRBCs. A combination light scattering information andfluorescence information is then used to further separate nRBCs fromWBCs. The systems and methods disclosed thereby ensure accurate countingand differentiation of nRBCs, WBCs, and WBC sub-populations. The systemsand methods also enable development of relatively milder WBC reagent(s),suitable for assays of samples containing fragile lymphocytes (or otherfragile WBCs), including aged samples.

In one embodiment, for example, the systems and methods disclosed hereininclude: (a) staining a blood sample with an exclusive cell membranepermeable fluorescent dye; (b) using a fluorescence trigger to screenthe blood sample for nuclei-containing particles; and (c) usingmeasurements of light scatter and fluorescence emission to distinguishnRBCs from WBCs. Systems and methods in accordance with the presentinvention show great advantages over traditional methods because theinterference from fragments of RBCs is substantially eliminated, and theresults of the assay become much less sensitive to the lysing strength.In other words, traditional nRBC analysis techniques are highlysensitive to lysing strength. If the strength of the lysing agent is tooweak, the fragments of RBCs or unlysed RBCs were also collected in theanalysis of WBCs. These fragments of RBCs or unlysed RBCs overlappednRBCs in many dimensions, resulting in difficulty in analyzing nRBCs. Onthe other hand, if the strength of the lysing agent was too strong (inorder to better deal with fragments of RBCs), a certain percentage oflymphocytes could be damaged and could be recognized as nRBCs.Therefore, the strength of the lysing agent is a problem in the analysisof WBCs. A key feature of the methods described herein is the reductionof interference from RBC fragments or unlysed RBCs in the analysis ofWBCs and nRBCs. Accordingly, variations in the strength of the lysingagent can be better tolerated. Even if the lysing agent is weaker thanusual, the unlysed RBCs do not interfere with the assay for nRBCs.

In the systems and methods described herein, quantification andidentification of nRBCs and WBCs can be obtained simultaneously in asingle assay. Alternatively, in the systems and methods describedherein, quantification and identification of nRBCs can be carried outwithout analysis of WBCs.

(1) Use of Fluorescent Dye(s).

WBCs and nRBCs contain a relatively high concentration of DNA in theirnuclei. Mature RBCs, however, do not contain DNA. Therefore, afluorescent dye is selected to differentiate two classes of blood cells;namely, the blood cells containing nucleic acids and the blood cells notcontaining nucleic acids. The purpose of the dye is to penetrate intolive cells easily, bind DNA with high affinity, and emit strongfluorescence with adequate Stokes shift when the dye is excited by anappropriate source of light. The peak absorption of the dye in thevisible band substantially matches the wavelength of the source of light(within 50 nm of the wavelength of the source of light, more preferably,within 25 nm of the wavelength of the source of light), in order to beproperly excite the dye and achieve optimal results.

The fluorescent dye selected is preferably: 1) capable of bindingnucleic acids, 2) capable of penetrating cell membranes of WBCs andnRBCs, 3) excitable at a selected wavelength when subjected to a sourceof light, 4) emits fluorescence upon excitation by the source of light,and 5) is biostable and soluble in a liquid. The dye may be selectedfrom group consisting of: acridine orange, SYBR 11, SYBR Green seriesdye, hexidium iodide, SYTO 11, SYTO 12, SYTO 13, SYTO 14, SYTO 16, SYTO21, SYTO RNA Select, SYTO 24, SYTO 25 and any equivalents thereof. Thedye is used to “activate” WBCs and nRBCs, and “screen out” unlysed RBCsand fragments of RBCs based on a fluorescence trigger configured in thehematology analyzer. The dye is typically present at a concentration offrom about 0.1 ng/mL to about 0.1 mg/mL. While various dyes areavailable, the dye selected is generally paired with the excitationsource of the hematology analyzer such that a single exclusive dye isused to stain and excite fluorescence emission in nRBCs and all WBCsub-populations intended to be identified, quantified, and/or analyzed.As such, a single (i.e., exclusive) dye can be used to identify,quantify, and analyze nRBCs and all the WBC subpopulations at once.

In one embodiment, a fluorescent dye is provided in a reagent, withcombinations of 1) at least one surfactant, 2) at least one buffer, 3)at least one salt, and/or 4) at least antimicrobial agent, in sufficientquantities for carrying out staining and activating up to 1,000×10³cells per microliter. The at least one surfactant, such as “TRITON”X-100 or saponin, is used to destroy the membranes of RBC, and reducethe sizes of fragments of RBCs. The at least one surfactant is typicallypresent at a concentration of from about 0.001% to about 5%. The atleast one antimicrobial agent, such as those from “TRIADINE” or“PROCLIN” families, is used to prevent the contamination of the reagentfrom microbes. The concentration of the at least one antimicrobial agentis sufficient to preserve the reagent for the shelf life required. Theat least one buffer, such as phosphate buffered saline (PBS) or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), is used toadjust the pH of reaction mixture for controlling lysis of RBCs andpreserving WBCs. The at least one buffer is typically present at aconcentration of from about 0.01% to about 3%. The pH typically rangesfrom about 3 to about 12. The at least one salt, such as NaCl or Na₂SO₄,is used to adjust the osmolality to increase the effect of lysing and/oroptimize WBC preservation. The at least one salt may be present at aconcentration of from about 0.01% to about 3%. In certain cases, the atleast one buffer can serve as the at least one salt, or the at least onesalt can serve as the at least one buffer. In general, lower osmolality,or hypotonicity, is used to accelerate the lysis of RBCs. The osmolalitytypically ranges from about 20 to about 250 mOsm.

Lysis of RBCs can be made to occur at a temperature above roomtemperature (e.g., between about 30° C. to about 50° C., such as about40° C.) over a relatively short period of time (e.g., less than about 25seconds, less than about 17 seconds, or even less than about 9 seconds),following mixing of the sample of blood and the reagent at a ratio ofabout one part by volume sample to about 35 parts by volume reagent. Thedata for analysis is collected with a plurality of optical channels andat least one fluorescence channel.

FIGS. 1A-1D show histograms of a sample of whole blood, showing theseparation of WBCs, nRBCs, and residues of RBCs following lysis, basedon optical and fluorescence measurements. More specifically, FIG. 1A isa histogram showing separation of particles based on measurements ofaxial light loss (ALL). FIG. 1B is a histogram showing separation ofparticles based on measurements of intermediate angle scatter (IAS).FIG. 1C is a histogram showing separation of particles based onmeasurements of 90° polarized side scatter (PSS). FIG. 1D is a histogramshowing separation of particles based on measurements of fluorescence(FL1). In the histograms, the horizontal axis indicates the value of thedetection channel (or the names of the channels, i.e., ALL, IAS, PSS, orFL1). The vertical axis indicates counts of components of the sample ofblood. In the histograms, the lines 100 indicate residues of RBCs, lines200 indicate WBCs, and lines 300 indicate nRBCs. As shown by comparingFIG. 1D to FIGS. 1A-1C, fluorescence information, rather than opticalmeasurements, shows much better separation between the nuclei-containingparticles (e.g., WBCs and nRBCs) and non-nuclei-containing particles(residues of RBCs). As used herein, “residues of RBCs” is synonymouswith “fragments of RBCs.”

(2) Use of a Fluorescence Trigger.

Blood cells emit different magnitudes of fluorescence signals uponexcitation of the fluorescent dye by a source of light. The differencesin magnitude of fluorescence signals arise from the quantity of nucleicacids, namely DNA, inside the cells. The greater the quantity of DNA,the greater the likelihood of higher fluorescence signals. Also,efficacy of penetration of cell membranes, size of the dye, bindingkinetics between the dye and DNA, affinity between the dye and DNA, andother factors, affect the fluorescence signals. Mature RBCs emit minimalfluorescence signals because there is no DNA within mature RBCs. nRBCsemit very strong fluorescence signals, because not only is DNA insidenuclei of nRBCs, but also the staining is easier because membranes ofnRBCs are destroyed during the lysis procedure. Unlysed RBCs or RBCfragments do not emit fluorescence, although they may emit very weakauto-fluorescence. As shown with reference to FIG. 1D, the cells thatemit much stronger fluorescence signals are the cells having nuclei,namely, all WBCs and nRBCs (when present).

As such, the systems and methods presented herein use a fluorescencetrigger for collecting and analyzing WBCs and nRBC. For example, afluorescence trigger, usually set between signals from RBCs and signalsfrom WBCs and nRBCs, can be used to collect signals from WBCs and nRBCsfor subsequent analysis.

(3) Use of a Plurality of Optical Channels and at Least One FluorescenceChannel for Analysis.

As used herein, the expression “fluorescence information” means datacollected from a fluorescence channel of a hematology analyzer. As usedherein, the expression “fluorescence channel” means a detection device,such as a photomultiplier tube, set at an appropriate wavelength bandfor measuring the quantity of fluorescence emitted from a sample.

In one embodiment, the blood sample analysis is conducted by means ofMultiple Angle Polarized Scattering Separation technology (MAPSS), withenhancement from fluorescence information. At least one photodiode, orat least one photomultiplier tube, or both at least one photodiode andat least one photomultiplier tube, are needed to detect light scatteredby each blood cell passing through a flow cell. Two or more photodiodesare used for measuring ALL signals, which measure about 0° scatter, andIAS signals, which measure low angle (e.g., about 3° to about) 15°scatter. Two or more photomultiplier tubes are used for detecting 90°polarized side scatter (PSS) signals and 90° depolarized side scatter(DSS) signals. Additional photomultiplier tubes are needed for FL1measurements within appropriate wavelength range(s), depending on thechoice of wavelength of the source of light. Each event captured on thesystem thus exhibits a plurality of dimensions of information, such asALL, IAS (one or more channels), PSS, DSS, and fluorescence (one or morechannels). The information from these detection channels is used forfurther analysis of blood cells.

FIG. 2 is a schematic diagram illustrating the illumination anddetection optics of an apparatus suitable for hematology analysis(including flow cytometry). Referring now to FIG. 2, an apparatus 10comprises a source of light 12, a front minor 14 and a rear mirror 16for beam bending, a beam expander module 18 containing a firstcylindrical lens 20 and a second cylindrical lens 22, a focusing lens24, a fine beam adjuster 26, a flow cell 28, a forward scatter lens 30,a bulls-eye detector 32, a first photomultiplier tube 34, a secondphotomultiplier tube 36, and a third photomultiplier tube 38. Thebulls-eye detector 32 has an inner detector 32 a for 0° light scatterand an outer detector 32 b for 7° light scatter.

In the discussion that follows, the source of light is preferably alaser. However, other sources of light can be used, such as, forexample, lamps (e.g., mercury, xenon). The source of light 12 can be avertically polarized air-cooled Coherent Cube laser, commerciallyavailable from Coherent, Inc., Santa Clara, Calif. Lasers havingwavelengths ranging from 350 nm to 700 nm can be used. Operatingconditions for the laser are substantially similar to those of laserscurrently used with “CELL-DYN” automated hematology analyzers.

Additional details relating to the flow cell, the lenses, the focusinglens, the fine-beam adjust mechanism and the laser focusing lens can befound in U.S. Pat. No. 5,631,165, incorporated herein by reference,particularly at column 41, line 32 through column 43, line 11. Theforward optical path system shown in FIG. 2 includes a sphericalplano-convex lens 30 and a two-element photo-diode detector 32 locatedin the back focal plane of the lens. In this configuration, each pointwithin the two-element photodiode detector 32 maps to a specificcollection angle of light from cells moving through the flow cell 28.The detector 32 can be a bulls-eye detector capable of detecting axiallight loss (ALL) and intermediate angle forward scatter (IAS). U.S. Pat.No. 5,631,165 describes various alternatives to this detector at column43, lines 12-52.

The first photomultiplier tube 34 (PMT1) measures depolarized sidescatter (DSS). The second photomultiplier tube 36 (PMT2) measurespolarized side scatter (PSS), and the third photomultiplier tube 38(PMT3) measures fluorescence emission from 440 nm to 680 nm, dependingupon the fluorescent dye selected and the source of light employed. Thephotomultiplier tube collects fluorescent signals in a broad range ofwavelengths in order to increase the strength of the signal.Side-scatter and fluorescent emissions are directed to thesephotomultiplier tubes by dichroic beam splitters 40 and 42, whichtransmit and reflect efficiently at the required wavelengths to enableefficient detection. U.S. Pat. No. 5,631,165 describes variousadditional details relating to the photomultiplier tubes at column 43,line 53 though column 44, line 4.

Sensitivity is enhanced at photomultiplier tubes 34, 36, and 38, whenmeasuring fluorescence, by using an immersion collection system. Theimmersion collection system is one that optically couples the first lens30 to the flow cell 28 by means of a refractive index matching layer,enabling collection of light over a wide angle. U.S. Pat. No. 5,631,165describes various additional details of this optical system at column44, lines 5-31.

The condenser 44 is an optical lens system with aberration correctionsufficient for diffraction limited imaging used in high resolutionmicroscopy. U.S. Pat. No. 5,631,165 describes various additional detailsof this optical system at column 44, lines 32-60.

The functions of other components shown in FIG. 2, i.e., a slit 46, afield lens 48, and a second slit 50, are described in U.S. Pat. No.5,631,165, at column 44, line 63 through column 45, line 26. Opticalfilters 52 or 56 and a polarizer 52 or 56, which are inserted into thelight paths of the photomultiplier tubes to change the wavelength or thepolarization or both the wavelength and the polarization of the detectedlight, are also described in U.S. Pat. No. 5,631,165, at column 44, line63 through column 45, line 26. Optical filters that are suitable for useherein include band-pass filters and long-pass filters.

The photomultiplier tubes 34, 36, and 38 detect either side-scatter(light scattered in a cone whose axis is approximately perpendicular tothe incident laser beam) or fluorescence (light emitted from the cellsat a different wavelength from that of the incident laser beam).

While select portions of U.S. Pat. No. 5,631,165 are referenced above,U.S. Pat. No. 5,631,165 is incorporated herein by reference in itsentirety.

The optical and fluorescence information collected may then be used todistinguish (or differentiate) nRBCs from WBCs (and WBCsub-populations). For example, two-dimensional cytograms (e.g.,cytograms showing PSS vs. ALL; ALL vs. IAS; and/or FL1 vs. ALL/IAS/PSS)can be used to identify and distinguish particles.

FIGS. 3A-3F are cytograms illustrating an analysis of a sample of bloodcontaining nRBCs at a % NRBC of 75%. More specifically, FIG. 3A is acytogram depicting axial light loss vs. intermediate angle scatter. FIG.3B is a cytogram depicting 90° polarized side scatter vs. axial lightloss. FIG. 3C is a cytogram depicting 90° polarized side scatter vs.intermediate angle scatter. FIG. 3D is a cytogram depicting FL1 vs.axial light loss. FIG. 3E is a cytogram depicting FL1 vs. intermediateangle scatter. FIG. 3F is a cytogram depicting FL1 vs. 90° polarizedside scatter. The %NRBC values obtained from the cytograms were 80.7 (asdetermined by manual gating using PSS v. ALL) and 76.3 (as determined byauto-clustering analysis carried out with MATLAB software).

FIGS. 4A-4F are cytograms illustrating an analysis of a sample of bloodcontaining nRBCs as a % NRBC of 1.0%. More specifically, FIG. 4A is acytogram depicting axial light loss vs. intermediate angle scatter. FIG.4B is a cytogram depicting 90° polarized side scatter vs. axial lightloss. FIG. 4C is a cytogram depicting 90° polarized side scatter vs.intermediate angle scatter. FIG. 4D is a cytogram depicting FL1 vs.axial light loss. FIG. 4E is a cytogram depicting FL1 vs. intermediateangle scatter. FIG. 4F is a cytogram depicting FL1 vs. 90° polarizedside scatter. The % NRBC values obtained from the cytograms were 1.2 (asdetermined by manual gating using PSS v. ALL) and 1.2 (as determined byauto-clustering analysis carried out with MATLAB software).

In a study using a total of 136 samples containing nRBCs, both manualmicroscope reviews and “CELL-DYN” Sapphire™ hematology analyzer resultswere used as reference for quantifications of nRBCs. Acridine orange, ata concentration of 3 μg/mL, was included in the a reagent. The sample ofblood and the reagent were mixed at a ratio of one part by volume sampleto 35 parts by volume of reagent. The mixture was incubated for a periodof 25 seconds at a temperature of 40° C. A sample measurement durationof 9 seconds was applied, with FL1 used as the sole trigger.Measurements of ALL, IAS, PSS, and FL1 were collected for each sample.The data was analyzed using both manual gating (PSS vs. ALL, or PSS vs.IAS) and auto-clustering analysis using MATLAB software. Thecorrelations of % nRBC between the results from the method of thepresent invention, and reference results were reported as shown in FIGS.5-8.

FIG. 5, for example, compares results (obtained by manual gating)against reference results, wherein the reference results were obtainedby a microscope review. As shown, the systems and methods of the presentinvention produced results in accordance with the slope and R² formulaof: Y=1.0404X; (R²=0.968).

FIG. 6 compares results (obtained by manual gating) against referenceresults, wherein the reference results were obtained by a “CELL-DYN”Sapphire™ hematology analyzer. As shown, the systems and methods of thepresent invention produced results in accordance with the slope and R²formula of: Y=1.1004X; (R²=0.956).

FIG. 7 compares results (obtained by auto-clustering) against referenceresults, wherein the reference results were obtained by a microscopereview. As shown, the systems and methods of the present inventionproduced results in accordance with the slope and R² formula of:Y=1.0215X; (R²=0.963).

FIG. 8 compares results (obtained by auto-clustering) against referenceresults, wherein the reference results were obtained by a “CELL-DYN”Sapphire™ hematology analyzer. As shown, the systems and methods of thepresent invention produced results in accordance with the slope and R²formula of: Y=1.0800X (R²=0.950)

Bhattacharyya distances (BD) between nRBCs and lymphocytes were alsocalculated based upon the mean positions of the clusters anddistribution coefficients of variation. The cluster separation isconsidered to be “good” if BD exceeds 3 and is considered to be“acceptable” if BD is greater than 2 and less than or equal to 3. Theaverage BD value for the tests were 4.64±1.89, ranging from 2.10 to11.95, for the 136 samples. Good separations (in which BD exceeds 3)between nRBCs and lymphocytes were observed in more than 85% (116/136)of samples containing nRBCs.

In contrast to the difficulty of balancing “over-lysing” and“under-lysing” for traditional methods of measuring nRBCs, the methodsdescribed herein preserve WBCs and provide the best separation betweennRBCs and lymphocytes (the WBC sub-population closest to nRBCs).Further, interference from fragments of RBCs is substantiallyeliminated.

Additional Embodiments

In another embodiment, there is provided a hematology analyzer forconducting a nucleated red blood cell (nRBC) analysis on a blood samplethat has been dyed with a fluorescent dye, wherein the fluorescent dyeis cell membrane permeable and nucleic acid binding. The analyzercomprises an excitation source positioned to excite particles within theblood sample. The analyzer also comprises a plurality of detectorsincluding: (1) an axial light loss detector positioned to measure axiallight loss from the excited blood sample, (2) an intermediate anglescatter detector positioned to measure intermediate angle scatter fromthe excited blood sample, (3) a side scatter detector positioned tomeasure 90° side scatter from the excited blood sample, and (4) afluorescence detector positioned to measure fluorescence emitted fromthe excited blood sample. The analyzer also comprises a processorconfigured to: (a) receive the measurements of (1) axial light loss, (2)intermediate angle scatter, (3) 90° side scatter, and (4) fluorescencefrom the plurality of detectors, and (b) perform a nRBC differentialanalysis of the blood sample, based on all four measurements, forparticles that emit fluorescence above a fluorescence threshold. Theside scatter detector may be a polarized side scatter detectorpositioned to measure 90° polarized side scatter from the excited bloodsample. The hematology analyzer may further comprise a depolarized sidescatter detector positioned to measure 90° depolarized side scatter fromthe excited blood sample. The processor may be further configured topre-screen the received measurements to remove from consideration anyparticles that do not meet the fluorescence threshold. The axial lightloss detector can measure axial light loss at 0° scatter. Theintermediate angle scatter detector can measure light angle scatter atabout 3° to about 15°. The plurality of detectors can include one ormore photomultiplier tubes. The excitation source may be a laserconfigured to emit light at a wavelength corresponding to thefluorescent dye. The fluorescent dye can be selected to correspond withthe excitation source.

The hematology analyzer may further comprise an incubation subsystem fordiluting the blood sample with a reagent. The reagent can include thefluorescent dye and a lysing agent. The reagent may alternativelycomprise (a) at least one surfactant, (b) at least one buffer or atleast one salt, (c) at least one antimicrobial agent, and (d) thefluorescent dye. The incubation subsystem may be configured to incubatethe blood sample with the reagent for a period of time of less thanabout 25 seconds, less than about 17 seconds, and/or less than about 9seconds. The incubation subsystem may be configured to incubate theblood sample with the reagent at a temperature ranging from about 30° C.to about 50° C., such as a temperature of about 40° C.

In another embodiment, there is provided a method of performing anucleated red blood cell nRBC analysis with an automated hematologyanalyzer. The method comprises the steps of: (a) diluting a sample ofwhole blood with a reagent, wherein the reagent includes a red bloodcells (RBC) lysing agent and a cell membrane permeable, nucleic acidbinding fluorescent dye; (b) incubating the diluted blood sample of step(a) for an incubation period of less than about 25 seconds, at atemperature ranging from about 30° C. to about 50° C.; (c) deliveringthe incubated sample from step (b) to a flow cell in the hematologyanalyzer; (d) exciting the incubated sample from step (c) with anexcitation source as the incubated sample traverses the flow cell; (e)collecting a plurality of light scatter signals and a fluorescenceemission signal from the excited sample; and (f) performing a nRBCanalysis based on all the signals collected in step (e), while removingfrom consideration any particles within the diluted blood sample that donot meet a fluorescence threshold based on the fluorescence emissionsignal.

The reagent may include: (a) at least one surfactant, (b) at least onebuffer or at least one salt, (c) at least one antimicrobial agent, and(d) at least one fluorescent dye. The excitation source may have awavelength of from about 350 nm to about 700 nm. The fluorescenceemission may be collected at a wavelength of from about 360 nm to about750 nm, by a band-pass filter or a long-pass filter. The plurality oflight scatter signals may include: (1) axial light loss, (2)intermediate angle scatter, and (3) 90° side scatter. The plurality oflight scatter signals may include: (1) axial light loss, (2)intermediate angle scatter, (3) 90° polarized side scatter, and (4) 90°depolarized side scatter.

In yet another embodiment, there are provided systems and methods forcounting nRBCs by means of an automated hematology analyzer. The systemsand method include means for and the steps of: (a) diluting a wholeblood sample containing nucleated red blood cells with at least onewhite blood cell reagent; (b) incubating the diluted sample of step (a)for a sufficient period of time within a selected temperature range tolyse red blood cells, allow nuclei of nucleated red blood cells to beexposed to the at least one white blood cell reagent, and allow at leastone fluorescent dye to stain nuclei of nucleated red blood cells, andpreserve white blood cells; (c) delivering the incubated sample of step(b) to a flow cell as a stream; (d) exciting the incubated sample with asource of light as the incubated sample traverses the flow cell; (e)collecting a plurality of optical scatter signals and at least onefluorescence emission signal simultaneously; and (f) differentiating andquantifying nucleated red blood cells by means of the opticalinformation and fluorescence information collected in step (e). Thesystems and methods further include: (1) the use of at least onefluorescent dye to bind and stain nucleic acids in WBCs and the nucleiof nRBCs in a given sample of blood during the procedure for lysingRBCs, and to induce fluorescence emissions upon being excited by photonsfrom a given source of light, such as a laser beam at an appropriatewavelength; (2) the use of a fluorescent trigger to separate and collectevents that emit strong fluorescence (i.e., WBCs and nRBCs); and (3) theuse of a plurality of optical channels and at least one fluorescentchannel for collecting and analyzing data in order to identify nRBCs,and to separate nRBCs from WBCs. The systems and method described hereinallows simultaneous analysis of WBCs and nRBCs. No additional reagent,preparation of samples, or analytical procedure is needed. Therefore,the method is efficient, cost-effective, and practical for moderndiagnostic use.

In one embodiment, the invention is directed towards one or morecomputer systems capable of carrying out the functionality describedherein. For example, any of the method/analysis steps discussed hereinmay be implemented in a computer system having one or more processors, adata communication infrastructure (e.g., a communications bus,cross-over bar, or network), a display interface, and/or a storage ormemory unit. The storage or memory unit may include computer-readablestorage medium with instructions (e.g., control logic or software) that,when executed, cause the processor(s) to perform one or more of thefunctions described herein. The terms “computer-readable storagemedium,” “computer program medium,” and “computer usable medium” areused to generally refer to media such as a removable storage drive,removable storage units, data transmitted via a communicationsinterface, and/or a hard disk installed in a hard disk drive. Suchcomputer program products provide computer software, instructions,and/or data to a computer system, which also serve to transform thecomputer system from a general purpose computer into a special purposecomputer programmed to perform the particular functions describedherein. Where appropriate, the processor, associated components, andequivalent systems and sub-systems thus serve as examples of “means for”performing select operations and functions. Such “means for” performingselect operations and functions also serve to transform a generalpurpose computer into a special purpose computer programmed to performsaid select operations and functions.

Conclusion

The foregoing description of the invention has been presented forpurposes of illustration and description. It is not intended to beexhaustive or to limit the invention to the precise form disclosed.Other modifications and variations may be possible in light of the aboveteachings. The embodiments were chosen and described in order to bestexplain the principles of the invention and its practical application,and to thereby enable others skilled in the art to best utilize theinvention in various embodiments and various modifications as are suitedto the particular use contemplated. It is intended that the appendedclaims be construed to include other alternative embodiments of theinvention; including equivalent structures, components, methods, andmeans.

The above Detailed Description refers to the accompanying drawings thatillustrate one or more exemplary embodiments. Other embodiments arepossible. Modifications may be made to the embodiment described withoutdeparting from the spirit and scope of the present invention. Therefore,the Detailed Description is not meant to be limiting. Further, theSummary and Abstract sections may set forth one or more, but not allexemplary embodiments of the present invention as contemplated by theinventor(s), and thus, are not intended to limit the present inventionand the appended claims in any way.

1. A hematology analyzer for conducting a nucleated red blood cell(nRBC) analysis on a blood sample that has been dyed with a fluorescentdye, wherein the fluorescent dye is cell membrane permeable and nucleicacid binding, the analyzer comprising: an excitation source positionedto excite particles within the blood sample; a plurality of detectorsincluding (1) an axial light loss detector positioned to measure axiallight loss from the excited blood sample, (2) an intermediate anglescatter detector positioned to measure intermediate angle scatter fromthe excited blood sample, (3) a side scatter detector positioned tomeasure 90° side scatter from the excited blood sample, and (4) afluorescence detector positioned to measure fluorescence emitted fromthe excited blood sample; and a processor configured to (a) receive themeasurements of (1) axial light loss, (2) intermediate angle scatter,(3) 90° side scatter, and (4) fluorescence from the plurality ofdetectors, and (b) perform a nRBC differential analysis of the bloodsample, based on all four measurements, for particles that emitfluorescence above a fluorescence threshold.
 2. The hematology analyzerof claim 1, wherein the side scatter detector is a polarized sidescatter detector positioned to measure 90° polarized side scatter fromthe excited blood sample.
 3. The hematology analyzer of claim 2, furthercomprising: a depolarized side scatter detector positioned to measure90° depolarized side scatter from the excited blood sample.
 4. Thehematology analyzer of claim 1, wherein the processor is furtherconfigured to pre-screen the received measurements to remove fromconsideration any particles that do not meet the fluorescence threshold.5. The hematology analyzer of claim 1, wherein the axial light lossdetector measures axial light loss at 0° scatter.
 6. The hematologyanalyzer of claim 1, wherein the intermediate angle scatter detectormeasures light angle scatter at about 3° to about 15°.
 7. The hematologyanalyzer of claim 1, wherein the plurality of detectors include one ormore photomultiplier tubes.
 8. The hematology analyzer of claim 1,wherein the excitation source is a laser.
 9. The hematology analyzer ofclaim 8, wherein the laser is configured to emit light at a wavelengthcorresponding to the fluorescent dye.
 10. The hematology analyzer ofclaim 1, wherein the fluorescent dye is selected to correspond with theexcitation source.
 11. The hematology analyzer of claim 1, furthercomprising: an incubation subsystem for diluting the blood sample with areagent.
 12. The hematology analyzer of claim 11, wherein the reagentincludes the fluorescent dye and a lysing agent.
 13. The hematologyanalyzer of claim 11, wherein the reagent includes (a) at least onesurfactant, (b) at least one buffer or at least one salt, (c) at leastone antimicrobial agent, and (d) the fluorescent dye.
 14. The hematologyanalyzer of claim 11, wherein the incubation subsystem is configured toincubate the blood sample with the reagent for a period of time of lessthan about 25 seconds.
 15. The hematology analyzer of claim 11, whereinthe incubation subsystem is configured to incubate the blood sample withthe reagent for a period of time of less than about 17 seconds.
 16. Thehematology analyzer of claim 11, wherein the incubation subsystem isconfigured to incubate the blood sample with the reagent for a period oftime of less than about 9 seconds.
 17. The hematology analyzer of claim11, wherein the incubation subsystem is configured to incubate the bloodsample with the reagent at a temperature ranging from about 30° C. toabout 50° C.
 18. The hematology analyzer of claim 11, wherein theincubation subsystem is configured to incubate the blood sample with thereagent at a temperature of about 40° C.
 19. A method of performing anucleated red blood cell nRBC analysis with an automated hematologyanalyzer, the method comprising: (a) diluting a sample of whole bloodwith a reagent, wherein the reagent includes a red blood cells (RBC)lysing agent and a cell membrane permeable, nucleic acid bindingfluorescent dye; (b) incubating the diluted blood sample of step (a) foran incubation period of less than about 25 seconds, at a temperatureranging from about 30° C. to about 50° C.; (c) delivering the incubatedsample from step (b) to a flow cell in the hematology analyzer; (d)exciting the incubated sample from step (c) with an excitation source asthe incubated sample traverses the flow cell; (e) collecting a pluralityof light scatter signals and a fluorescence emission signal from theexcited sample; and (f) performing a nRBC analysis based on all thesignals collected in step (e), while removing from consideration anyparticles within the diluted blood sample that do not meet afluorescence threshold based on the fluorescence emission signal. 20.The method of claim 19, wherein the reagent includes (a) at least onesurfactant, (b) at least one buffer or at least one salt, (c) at leastone antimicrobial agent, and (d) at least one fluorescent dye.
 21. Themethod of claim 19, wherein the excitation source has a wavelength offrom about 350 nm to about 700 nm.
 22. The method of claim 19, whereinfluorescence emission is collected at a wavelength of from about 360 nmto about 750 nm, by a band-pass filter or a long-pass filter.
 23. Themethod of claim 19, wherein the plurality of light scatter signalsinclude: (1) axial light loss, (2) intermediate angle scatter, and (3)90° side scatter.
 24. The method of claim 19, wherein the plurality oflight scatter signals include: (1) axial light loss, (2) intermediateangle scatter, (3) 90° polarized side scatter, and (4) 90° depolarizedside scatter.